Vasculogenesis, the development of new blood vessels, begins during the second week of normal human embryogenesis. Vascular development requires the tightly coordinated expression of several growth factors and their receptors. Among these are the Tie1 and Tie2 receptors which are almost exclusively endothelial cell specific. The critical transcriptional regulators of vascular-specific gene expression remain largely unknown.
Vascular development involves a complicated series of events including stem cell differentiation in developing embryos, endothelial cell interactions, and endothelial cell proliferation in developing tissue and in pre-existing endothelial cells, e.g., in angiogenesis. In an example of endothelial cell interaction, blood vessel development requires endothelial cells interact with surrounding mesenchymal cells for proper vessel development.
Angiogenesis may have beneficial or deleterious effect, depending on the circumstances in which is arises. For example, angiogenesis is a critical component of a number of diseases, in which it can have either beneficial or deleterious effects. The generation of new capillaries is necessary for normal healing in wound repair. In acute and chronic coronary ischemia the development of collateral blood vessels is a beneficial effect of angiogenesis. Examples of harmful effects of angiogenesis include neovascularization which results in diabetic retinopathy, and angiogenesis-dependent growth of many tumors.
Angiogenesis begins when clusters of endothelial cells fuse into cellular chords and eventually tubes, thus creating the new blood vessel. This process recapitulates the events that occur during embryonic blood vessel development. There has been considerable interest in identifying factors that regulate blood vessel development. Several growth factors have received a great deal of attention as regulators of endothelial cell differentiation and angiogenesis. An angiogenic growth factor that is highly expressed during embryogenesis, and appears to have unique target cell specificity for vascular endothelium is vascular endothelial growth factor (VEGF). Furthermore, its receptors Flk-1 and Flt-1 are expressed on the surface of developing and mature mouse blood vessels.
Tie1 and Tie2 are another family of endothelial-specific receptor tyrosine kinases which have been determined to be critical for vascular development. (Sato, T. N., et al., 1993. Proc Natl Acad Sci USA 90:9355-8). They are expressed predominantly on endothelial cells of the developing vasculature. Targeted disruption of Tie1 leads to the development of leaky blood vessels resulting in edema and hemorrhage, while disruption of Tie2 leads to dilated blood vessels and abnormal capillary networks, and early embryonic death (Sato, T. N., et al., 1995. Nature 376:70-4). The growth factor ligand for the Tie2 receptor, angiopoietin-1, has been recently identified (Davis, S., et al., 1996. Cell 87:1161-9). Mutations in the Tie2 gene have been identified in humans, resulting in venous malformations (Vikkula, M., et al., 1996. Cell 87:1181-90). Tie1 and Tie2 gene expression has been shown to be upregulated during tumor angiogenesis (Hatva, E., et al., 1995. Am J Pathol 146:368-78; Kaipainen, A., et al., 1994. Cancer Res 54:6571-7). Although these receptors were described as being completely endothelial cell specific it has recently been shown that both receptors are expressed in up to 30 percent of undifferentiated hematopoietic stem cells and 10 percent of B cells, suggesting a possible role in hematopoiesis in addition to vasculogenesis (Hashiyama M, et al. Blood. 1996;87:93-101; Yano M, et al. Blood. 1997;89:4317-4326).
Although much information has emerged concerning the possible role of growth factors and their receptors during vascular development, little is known about the nuclear events that orchestrate this process at the transcriptional level.
The Ets genes are a family of at least thirty members that function as transcription factors and play a central role in regulating genes involved in development, cellular differentiation and proliferation. Interestingly, the main regulatory elements of the Flt-1, Tie1, and Tie2 genes have several conserved putative Ets binding sites, which are critical for the transcriptional activity of the promoters and enhancers of these genes. For example, a mutation of one Ets binding site in the promoter of the Flt-1 gene leads to a ninety-percent reduction in the basal activity of the promoter. Likewise, in transgenic animals in which LacZ expression is directed throughout the vasculature by the Tie2 promoter and enhancer, a mutation in an Ets binding site in the core enhancer leads to a marked reduction in vascular directed LacZ gene expression. It is currently unknown, which of the Ets factors are critical for the transcriptional activity of these genes.
Thus, it would be useful to be able to regulate blood vessel development and/or endothelial cell differentiation in order to treat certain types of diseases that involve vascular development. For example, it would be useful to block blood vessel development, i.e., angiogenesis, in diseases such as certain cancers, diabetic retinopathy and inflammation, e.g., rheumatoid arthritis. Currently, however, the ability to block angiogenesis in a variety of diseases remains incomplete. The major shortcomings of the currently available antiangiogenesis drugs are that they may not completely block angiogenesis. The identification of novel angiogenesis inhibitors, especially those that offer a more complete means of blocking angiogenesis, is highly desirable.
Furthermore the ability to increase blood vessel development to treat certain diseases, or symptoms of certain diseases, is highly desirable. For example, known compounds that induce blood vessel development for treating certain diseases, e.g., in coronary heart disease and after myocardial infarction, are limited, and the discovery of small molecules or novel proteins that could enhance the ability to enhance angiogenesis in these disease states is also highly desirable.